Urolithiasis is a multi-factorial disease and it is unlikely that a single factor will be responsible for the entire spectrum of this disorder. Nonetheless, oxalate and calcium-oxalate crystal (COM-crystal) interactions with renal epithelial cells, and the cellular responses that follow are important, but poorly understood subjects in this disorder. Signal transduction mechanisms that mediate the effects of oxalate and COM-crystals in renal epithelial cells are critical determinants in nephrolithiasis and have been the primary focus of our investigation. During the five year funding period, we demonstrated that oxalate and COM-crystals activate p38 MAP kinase pathway and NF-kB transcription factor. We also observed that exposure of human renal epithelial cells to oxalate as well as COM-crystals results in increased production of IL-6, a pro-inflammatory cytokine, indicating generation of inflammatory signals. However, the mechanism of these actions remains unclear. The proposed studies will evaluate the hypothesis that p38 MAP kinase mediated NF-kB signaling plays central role in mediating the effects of oxalate and COM-crystals, and in modulating the cellular responses. This hypothesis is driven by our observations that oxalate and COM-crystal exposure caused a rapid and robust activation of p38 MAP kinase and NF-kB in renal epithelial cells. However, the precise sequence/s through which oxalate and COM-crystals cause NF-kB activation is not understood. The present studies have four objectives: (1) to elucidate the molecular mechanisms involved in oxalate/COM-crystal induced activation of NF-kB pathway;(2) to investigate the roles played by NF-kB pathway in mediating the effects of oxalate/ COM- crystals;(3) to evaluate the molecular mechanisms by which oxalate/ COM-crystals regulates IL-6 levels in renal epithelial cells with special emphasis on the role of p38 MAP kinase pathway and NF-kB transcription factor activation;and (4) to evaluate the role of p38 MAP kinase signaling and NF-kB activation in vivo animal models of hyperoxaluric nephrolithiasis. Understanding the specific signaling processes that mediate the effects of oxalate and COM crystals in renal epithelial cells will help us develop strategies to inhibit the pathologic effects of these agents and prevent crystal retention and renal stone formation. We anticipate that proposed studies together with our preliminary data will identify p38 MAP kinase pathway and NF-kB as a mechanism-based target/s for the prevention of crystal retention. As a translational approach, the long-range goal of these studies is to define and establish the usefulness of p38 MAP kinase and NF-kB inhibitors for the prevention and therapy of human kidney stone disease.PUBLIC HEALTH RELEVANCE: Kidney stone disease is a multifactorial disorder and it is unlikely that a single factor will be responsible for all the subsets of stone patients. Nonetheless, one important factor in genesis of stone disease is retention of crystalline materials in the renal tubules. Therefore, understanding the signaling mechanisms that mediate the effects of oxalate and COM-crystals following their interaction with renal tubular cells are essential in our understanding the molecular events associated with this disorder. We have obtained evidence indicating activation of p38 MAP kinase and NF-kB transcription factor in renal cells upon oxalate and COM-crystal interaction in vitro cell culture systems. The goal of the proposed studies is to further define this pathway using primary cultures of human kidney cells and to test the hypothesis that, "p38 MAP kinase pathway mediated NF-kB transcription factor plays a central role in mediating the effects of oxalate and COM- crystals by regulating gene expression". We also seek to extend these observations in current experimental animal models of the stone disease. Moreover, we will test feasibility of a pharmacological inhibitors of p38 MAP kinase and NF-kB in preventing oxalate and COM-crystal induced pathophysiological events in the experimental animals. Taken together our studies focus on characterizing p38 MAP kinase pathway and NF-kB as a molecular targets for intervention in urolithiasis. Thus this proposed study completely satisfies the requirements set forth in the Program Announcement.